STRUCTURAL REQUIREMENTS OF ANTIBODY COMBINING SITES

这项研究的长期目标是了解如何 抗体可变区的氨基酸序列导致抗体 对抗原和独特型表达的特异性和亲和力。 抗体基因的寡核苷酸定向突变将用于 在补体决定区(CDR)中引入突变 鼠抗对偶氮苯基砷酸(ARs)抗体。突变抗体 在将诱变基因导入到 骨髓瘤或杂交瘤细胞株，以及转基因瘤将被恢复 在液体微量培养或软琼脂中。分泌抗体，在 上清液或原位，将通过标准的免疫学分析进行筛选。 抗体亲和力对变化特别敏感的假设 在某些职位上将通过引入所有可能的 已发现影响的四个位置中的每一个的替换 抗ARS抗体的亲和力。改进的最低要求 抗体亲和力和改变的特异性将通过以下方式确定 在CDR中一次引入一个或几个随机突变 抗ARs抗体，筛选亲和力增强的突变体 ARS或具有新的抗原特异性。为了探索每个人的角色 抗ARs抗体的6个CDR在抗原结合和独特型中的作用 表达式中，CDR将被替换为 甘氨酸残留物。将重建一个反ARS结合点 然后尝试通过在这种甘氨酸取代的CDR中引入氨基 与Ars接触有关的酸性残留物。这个 甘氨酸取代的CDR也将在低频率下随机诱变， 以获得针对ARs或其他抗原的抗体。真知灼见 从拟议的研究中获得的成果应该会加深我们对 蛋白质的折叠，特别是抗体的互补性， 应该会让我们更接近裁剪的长期目标 用于研究和医疗目的的抗体。

The long term objective of this study is to attain an understanding of how the amino acid sequence of antibody variable regions leads to antibody specificity and affinity for antigen, and to idiotypic expression. Oligonucleotide-directed mutagenesis of antibody genes will be used to introduce mutations in the complementarity determining regions (CDRS) of a mouse antibody specific for p-azophenylarsonate (Ars). Mutant antibodies will be expressed following transfection of the mutagenized genes into myeloma or hybridoma cell lines, and transfectomas will be recovered either in liquid microcultures or in soft agarose. Secreted antibodies, in supernatants or in situ, will be screened by standard immunological assays. The hypothesis that antibody affinity is particularly sensitive to changes at certain positions will be tested by introducing all possible substitutions at each of four positions that have been found to affect affinity of the anti-Ars antibody. Minimal requirements for improved antibody affinity and for changed specificity will be determined by introducing one or a few random mutations at a time in the CDRs of the anti-Ars antibody, and screening for mutants with increased affinity for Ars or with new antigen specificities. To probe the roles of each of the six CDRs of the anti-Ars antibody in antigen binding and idiotypic expression, the CDRs will be individually "blanked out" by replacement with glycine residues. The reconstruction of an anti-Ars combining site will then be attempted by introducing into such glycine-replaced CDRS, the amino acid residues that have been implicated in Ars contact. The glycine-replaced CDRs will also be randomly mutagenized at low frequency, to obtain antibodies specific for Ars or for other antigens. Insights gained from the proposed studies should further our understanding of protein folding in general and of antibody complementarity in particular, and should bring us a step closer to the long term objective of tailoring antibodies for research and medical purposes.